A. Site-specific Mutagenesis of Presumptive ATP Binding Sites in Escherichia coli Adenylyl Cyclase. In addition to the region of the enzyme previously studied, two potential regions of interest were subjected to site-directed mutagenesis treatments. The focus of attention was on possibly essential lysine residues in these regions. It was found that the change of lysine 90 to either methionine or glutamine had a minor effect on the Vmax and that the Km for the glutamine mutant was reduced by a factor of 3. In contrast, the change of lysine 196 to methionine eliminated the activity and the change to glutamine reduced the activity by one order of magnitude and the Km by a factor of 3. B. Stimulation of Adenylyl Cyclase Activity by Nucleotides. Permeable cells, but not extracts, of E. coli contain adenylyl cyclase in a form that is stimulated by GTP as well as UTP and CTP. The kinetic properties of the enzyme are consistent with the interpretation that the nucleotides act as allosteric regulators of the activity. Mixing studies support the conclusion that there is a single site for all the active nucleotides. The expression of the allosteric stimulatory effect requires the presence of the proteins of the Phosphoenolpyruvate; sugar Phosphotransferase System. C. Involvement of Enzyme I of the Phosphoenolpyruvate: Sugar Phosphotransferase System as an Activator of Adenylyl Cyclase in E. coli. Enzyme IIIglc of the Phosphoenolpyruvate: sugar Phosphotransferase System has been concluded to be a positive regulator of adenylyl cyclase activity when it is in the phosphorylated form. Since Enzyme I of this phosphotransferase system is required to maintain Enzyme IIIglc in the phospho-form, it had been assumed that this protein acts only indirectly to activate adenylyl cyclase. A strategy was developed to allow Enzyme I to phosphorylate Enzyme IIIglc but not form a complex with the protein. Under these conditions, the phosphorylated form of Enzyme IIIglc did not stimulate adenylyl cyclase activity. The conclusion from these studies is that Enzyme I is actually an activator of adenylyl cyclase.